Objective: To determine the presence of zoonotic tick-borne bacteria in feral pigeons (Columba livia domestica) from urban areas. Methods: Spleen samples from 84 feral pigeons, found dead with traumatic injuries in urban areas, were examined by PCR to detect DNA of Anaplasma phagocytophilum, Bartonella spp., Borrelia burgdorferi sensu lato, Coxiella burnetii, Rickettsia spp., and Chlamydophila spp. Results: Twenty (23.8%) pigeons were infected by tick-borne agents, in particular 2 (2.38%) animals resulted positive for Bartonella spp., 5 (5.95%) for C. burnetii, 5 (5.95%) for Rickettsia spp., 13 (15.47%) for B. burgdorferi sensu lato. All birds scored negative for A. phagocytophilum. Moreover, 17 (20.23%) pigeons were positive for Chlamydophila spp. and among them 10 (11.9%) for Chlamydophila psittaci. Mixed infections by two or three agents were detected in 8 (9.52%) animals. Conclusions: Feral pigeons living in urban and periurban areas are a hazard for the human health as source of several pathogens. The obtained results confirm pigeons as reservoirs of chlamydial agents and suggest that they may be involved in the epidemiology of zoonotic tick-borne infections too.

Objective: To investigate the potential role of wild birds as fecal spreaders of enteropathogenic, enterohemorrhagic and Shiga-toxins producing Escherichia coli (E. coli), enteropathogenic E. coli, enterohemorrhagic E. coli and Shiga toxin-producing E. coli strains. Methods: Fecal samples collected from 121 wild birds of different orders and species were submitted to molecular analyses. In particular, eaeA encoding intimin, hlyA encoding for hemolysin, stx1 and stx2 genes encoding Shiga-toxins 1 and 2, respectively, were investigated. Results: Overall, 21(17.35%) fecal samples resulted positive for at least one of the investigated genes. In detail, 12(9.91%) samples were positive for eaeA, 10(8.26%) for stx1, 4(3.31%) for hylA and 1(0.83%) for stx2. An owl (Athene noctua) positive for the four investigated genes suggesting that it harbored a STEC strain. However, virulence genes characterizing EPEC, and EHEC strains were mainly found among seagulls, waterfowl and feral pigeons. Conclusions: Seagulls, waterfowl and feral pigeons, which frequently reach and contaminate rural, urban and peri-urban areas with their droppings, may be important sources of E. coli infection for other animals and humans.

Objective To investigate the occurrence of Bartonella sp. infection in asymptomatic horses and donkeys living in Tuscany, Central Italy. Methods Blood samples were collected from 77 horses and 15 donkeys and tested by indirect immunofluorescent test to detect antibodies against Bartonella sp. and by PCR to detect the pathogen. Results Fifty-four (58.69%; 95% CI: 47.95%–68.87%) animals, 9 donkeys and 45 horses, were seropositive with antibody titers ranging from 1:64 to 1:512. PCR assays detected 9 horses positive for Bartonella sp. and 3 donkeys for Bartonella henselae genotype I. Conclusions The detected sero-prevalence suggests a common and frequent exposure of equids living in Central Italy to bartonellae and PCR results show that Bartonella sp. infection is possible both in horses and donkeys. At the best of our knowledge, this is the first report of Bartonella henselae infection in donkeys.

Objective: To determine the prevalence of zoonotic tick-borne bacteria in feeding ticks removed from hunted wild animals. Methods: PCR was executed on DNA extracted from 77 tick pools to detect Anaplasma phagocytophilum, Bartonella spp., Borrelia burgdorferi sensu lato, Coxiella burnetii and Rickettsia spp. Results: A total of 432 ticks were collected: 30 (6.94%) Haemaphysalis punctata, 72 (16.7%) Dermacentor marginatus and 330 (76.38%) Ixodes ricinus. For each animal one or two pools of 3 ticks of the same species was constituted. Seventy-seven tick pools were examined by PCR: 58 (75.32%) resulted infected and among them 14 (18.18%) showed co-infections. In particular, 29 (37.66%) pools were positive for Bartonella spp., 23 (29.87%) for Anaplasma phagocytophilum, 16 (20.78%) for Rickettsia spp., and 5 (6.49%) for Borrelia burgdorferi s.l. All samples were negative for Coxiella burnetii. Conclusions: The results demonstrate the presence of several zoonotic tick-borne pathogens in the studied area, and underline the risk of exposure to infections for hunters not only during the outdoor activity, but also when they manipulate hunted animals infested by infected ticks.

Objective: To determine the prevalence of vector-borne bacteria and protozoa in hunting dogs living in Central Italy. Methods: Molecular testing was executed on DNA which was extracted from blood specimens collected from 117 asymptomatic dogs to detect Anaplasma phagocytophilum, Babesia canis (B. canis), Bartonella spp., Coxiella burnetii (C. burnetii), Ehrlichia canis, Hepatozoon canis, and Leishmania infantum. Results: A total of 48 dogs (41.0%) were infested by Ixodes ricinus and Rhipicephalus sanguineus ticks. Tick-borne infections were observed in 64 (54.7%) animals. More in detail, 38 dogs (32.5%) screened positive for Hepatozoon canis, 24 (20.5%) for Bartonella vinsonii subsp. berkhoffii, 20 (17.1%) for Leishmania infantum, 6 (5.1%) for C. burnetii, 5 (4.3%) for B. canis (3 B. canis vogeli and 2 B. canis canis), 3 (2.5%) for Anaplasma phagocytophilum, and 2 (1.7%) for Ehrlichia canis. Mixed infection by 2 agents occurred in 17 (14.5%) subjects, by 3 agents in 7 (6.0%) dogs, and by 4 agents in 1 (0.9%) animal. Conclusions: The results demonstrated that several vector-borne pathogens were circulating in this region and dogs infected by these agents were usually asymptomatic. A relevant finding was the presence of DNA of C. burnetii, a severe zoonotic agent, in the 5.1% of tested dogs, which can be source of infection for their owners not only through tick bites, but also directly with urine, feces and birth products.

Objective To determine the exposure of wild brown hares [Lepus europaeus (L. europaeus), pallas] to Anaplasma phagocytophilum (A. phagocytophilum), Borrelia burgdorferi (B. burgdorferi) sensu lato, Encephalitozoon cuniculi (E. cuniculi), Leishmania sp., Neospora caninum (N. caninum) and Toxoplasma gondii (T. gondii). Methods Two hundred twenty-two blood serum samples of wild brown hares captured in protected areas of the province of Pisa (Central Italy) were tested to detect antibodies against the reported pathogens. Results Thirty one (14.0%) animals resulted positive for at least one tested agent, with antibody titres ranging from 1:20 to 1:320. In particular, 13 (5.8%) samples were positive to B. burgdorferi s.l., 11 (4.9%) to N. caninum, 3 (1.3%) to T. gondii, 2 (0.9%) to A. phagocytophilum and 2 (0.9%) to Leishmania sp. No samples scored positive to E. cuniculi. Four animals (14.8%) resulted coinfected with 2 different pathogens. Conclusion The obtained results showed that B. burgdorferi s.l. N. caninum, T. gondii, A. phagocytophilum and Leishmania sp. circulate in wild brown hares in Central Italy, suggesting a possible role of L. europaeus as reservoir of these pathogens. The obtained results showed that autochthonous wild brown hares living in Central Italy have been exposed to several pathogens circulating in this area, suggesting a possible role of L. europaeus as reservoir.

Objective To investigate some pathogenic characters of Salmonella enterica strains isolated from poultry. Methods Twenty-three genetically distinct Salmonella enterica strains, of different serovars and pulsotype, were examined for virulence traits. Resistance to gastric acid environment was estimated by measuring the percentage of survived bacterial cells after exposure for 2 h to a synthetic gastric juice. Strains were analyzed with PCR for the presence of the following virulence genes: mgtC and rhuM located on SPI-3, sopB and pipB located on SPI-5, Salmonella virulence plasmid (spv) R (spvR), spvB and spvC located on Salmonella plasmid virulence and sodCI, sopE, and gipA located on prophage. Finally, resistance to 21 antibiotics was tested with Kirby–Bauer method. Results A percentage of 82.60% of strains were resistant to gastric environment after induction and 60.87% of the strains exhibited constitutive resistance too. Nineteen different virulence profiles were detected. The phage related genes sodCI and sopE and the plasmid mediated operon spvR, spvB and spvC (spvRBC) were detected in 82.60%, 47.82% and 52.17% of strains, respectively. Typhimurium and Enteritidis strains showed the highest number of virulence genes. Twenty-one different antibiotic resistance profiles were obtained and two isolates (Typhimurium and Enteritidis) resulted sensible to all the tested molecules. The ampicillin, streptomycin, sulfonamide and tetracycline resistance profile was detected in seven isolates (30.43%). Conclusion Our results show that paratyphoid Salmonella strains with several characters of pathogenicity, that may be cause of severe pathology in animals and humans, are circulating among poultry.

Objective To investigate clinicopathological, bacteriological and pathological aspects of an experimental infection with Yersinia pseudotuberculosis (Y. pseudotuberculosis) in hares to verify the efficacy of serology for the in vivo diagnosis. Moreover, the pathogenicity of two Y. pseudotuberculosis strains was investigated in order to detect potential differences. Methods Twelve European brown hares (Lepus europaeus, Pallas) were experimentally infected per os and via conjunctival mucosae with Y. pseudotuberculosis: six subjects were infected with a strain isolated from a naturally infected hare (YpH) and six subjects with a strain isolated from a naturally infected rabbit (YpR). Two hares were used as negative controls. All animals were subjected to clinical, bacteriological and serological examinations during 9 weeks following the infection and, at the end of the control period, subjects still alive were euthanized and submitted to a complete post mortem examination. Results All faecal samples collected during the control period were positive for bacteriological examinations and to a PCR for the inv gene of Y. pseudotuberculosis, while only one YpH-infected hare showed a positive haemocultures. From the 2nd to the 9th week post infection (pi), serological analysis revealed specific antibodies with titers ranging from 1:10 to 1:160 in all YpH-infected and two YpR-infected subjects. All the YpH-infected and two YpR-infected hares scored positive for Y. pseudotuberculosis by means of bacteriological investigations. Grossly, suppurative multifocal lesions were detected in liver, spleen, kidney and sub-mandibular lymph nodes in both YpH- and YpR-infected hares and confirmed with histopathology. Pulmonary lesions were observed only in YpH-infected subjects. Immunohistochemistry confirmed the presence of bacterial antigen in all infected animals. Conclusion Results of this study revealed that YpH strain is more pathogenic for hares than the YpR strain; moreover the serological test performed in this study could be used for the diagnosis of pseudotuberculosis in hares, whereas post mortem diagnosis should be confirmed by means of bacteriological examination, PCR, histopathology and immunohistochemistry.